Fabry disease: We extended our pre-clinical investigations on gene therapy for this hereditary metabolic disorder using the alpha-galactosidase A (alpha-Gal A) knock-out animal model of Fabry disease created by this Branch. We constructed an adeno-associated virus (AAV) vector containing the chicken alpha-actin promoter and the human alpha-Gal A gene. A single intravenous injection of this vector into Fabry mice caused long-term enzymatic and functional correction in multiple organs of these animals. Alpha-gal A activity in the liver, heart and spleen of the recipients became higher than that in wild-type mice of the same strain. Significant increases of alpha-Gal A activity also occurred in the kidney, lung and small intestine of the injected mice. Following injection of the vector, there was complete clearance of accumulating globotriaosylceramide (Gb3) in the liver, spleen and heart of the Fabry mice that lasted more than 24 weeks. Gb3 decreased in the lung and small intestine, but the levels were not completely restored to that of normal mice. Gb3 in the kidney was reduced to nearly normal by 8 weeks after injection of the vector. However, by 24 weeks after administering the vector, Gb3 had begun to reaccumulate in this organ. There were no signs of toxicity to AAV. These findings provide a realistic basis and an extraordinarily strong incentive to explore gene therapy in patients with Fabry disease. They further indicate that this technique merits examination for the treatment of many other hereditary disorders of metabolism. Delivery of Genes to the Central Nervous System: Many hereditary disorders are characterized by brain pathology and developmental abnormalities. We created a novel lentiviral vector construct to deliver therapeutic genes to the central nervous system. This vector has the ability to deliver required genes to non-dividing cells such as neurons in the brain. Moreover, we have engineered the vector to include the herpes simplex virus type 1 tegument protein VP-22. The addition of VP-22 facilitated the intercellular delivery of protein products of genes from the transduced cells to non-infected cells. This technique greatly enhances the effectiveness of gene therapy to the CNS. Many investigators have requested this vector from DMNB. We have also developed a lenti-virus based gene trap vector. This vector greatly facilitates the identification of important, but previously uncharacterized genes and their biological functions. It also has significant advantages for studies on cell differentiation and lineage commitment. In addition, it may prove useful for examining the function of specific gene products in vivo and their correct localization within the molecular circuitry of appropriate cell types. This construct has also been requested frequently by other investigators at NIH and by extramural scientists. Mucolipidosis IV: We previously reported the cloning of the gene that is mutated in patients with this disorder. This gene produces a protein called mucolipin. Mucolipin is a member of the TRP family of proteins. This discovery implies that the dominant pathophysiological alteration in mucolipidosis IV (MLIV) is a channelopathy. We are developing testable in vitro and in vivo models of this human neurogenetic disorder. The first of these investigations consists of electrophysiological studies in artificial lipid bilayers into which normal and mutated forms of mucolipin are inserted. This study revealed that ion channel dysfunction is produced by disease-related mutations in the MLIV gene. The second approach employs telomerase-immortalized skin fibroblasts derived from normal humans and from MLIV patients that contain specific point mutations in the MLIV gene. Studies with these cells provided additional insight into the ion channel properties of mucolipin. The third investigation involves selective silencing of mucolipin RNA to block expression of the MLIV gene in a continuous human parietal cell line. Experiments with this cellular model are expected to be extraordinarily significant since the major documented biochemical defect in MLIV is the complete absence of acid secretion by parietal cells. This investigation should lead to an understanding of the role of mucolipin in acid production. The fourth line of investigation centers on the creation of a murine model of MLIV. We have prepared an appropriately targeted gene construct, and we will begin blastocyst injections with it to create an MLIV knock-out mouse in the near future.